Introduction: The ability of COVID-19 immunization to stimulate immune responses in immunocompromised populations such as solid organ transplant (SOT) recipients is not well understood. Most research has focused on antibody responses, but there is growing recognition of the important role of vaccine-induced T cell responses as a requirement for protective immunity. Activation-induced marker (AIM) assays enable quantification of antigen-specific T cell responses by detection of antigen-stimulated cell surface marker upregulation. AIM assays do not require knowledge of HLA or epitopes and detect antigen-specific T cells independently from their cytokine profile or proliferative status, and thus represent an ideal method to evaluate T cell responses in patient cohorts. We developed an optimized AIM assay protocol to quantify vaccine-induced T cell immunity to SARS-CoV-2 spike (S) protein variants in a multi-centre, prospective clinical cohort study of COVID-19-vaccinated SOT recipients.
Methods: We tested various aspects of AIM assays to determine parameters for optimal sensitivity, specificity, reproducibility and feasibility. Inter- and intra-laboratory reproducibility of an optimized protocol was tested using replicate aliquots of PBMCs from healthy volunteers and measuring responses to CMV and the ancestral strain S protein. We are now assessing T cell responses upon 2 or 3 doses of COVID-19 vaccine in a cohort of SOT recipients.
Results: Twenty hours post-stimulation with Wuhan S protein peptide pools, the optimal cell surface markers to detect S-specific were CD25, CD69, CD134 and CD137 for CD4+ T cells and CD69, CD107a, and CD137 for CD8+ T cells. Representative data from healthy donor PBMCs are shown in Figure 1. Intra-site testing revealed batch-dependent effects, suggesting the need for careful design of experimental batches and testing of replicate aliquots in different batches. Variability could be reduced by excluding samples with less than 75% viability. Using this optimized AIM assay, we are currently analysing samples from the SOT cohort, which includes 33 kidney, lung or heart transplant recipients who received COVID-19 mRNA vaccines. PBMCs were collected following administration of the 2nd and 3rd dose, as well as 6 and 12 months post-1st dose. AIM-assay-based T cell responses to Wuhan- and BA.4/5 S protein peptides will be assessed at each timepoint post-vaccination, and statistical analysis will determine how T cell immunity may vary with vaccine brand, type of transplant and other parameters.

Conclusions: The optimized AIM assay protocol provides a critical tool to evaluate vaccine effectiveness. The ongoing clinical cohort study will demonstrate the applicability of the assay to immune-compromised SOT patients and will fill a gap in knowledge about how well mRNA vaccines induce T cell responses in this unique patient population.